NIH AREA (R15) Program Heart failure is a major public health problem and defined as a chronic and complex clinical syndrome which results from any structural or functional cardiac disorder that impairs the ability of the heart to efficietly pump blood. Understanding how perturbations in cardiac cell signaling leads to the development of heart failure is lacking, although many insightful studies have identified key signaling components involved in protein/gene expression, Ca2+ cycling, and post-translational modification. These complex cellular functions are controlled by a small number of second messengers, including Ca2+ and cAMP. The diversity of this complex network of cellular responses is mediated by second messengers and achieved in part, by scaffolding proteins. Scaffolding proteins assemble intracellular signaling components of a network cascade into a specific complex within the cell, thereby enhancing signaling specificity as well as enhancing signaling efficiency. A-Kinase Anchoring Proteins (AKAPs) is an example of scaffolding proteins that fine-tunes cellular responses - cellular proximity and protein effective concentrations - by forming multi-component protein complexes. This project will focus on one particular AKAP, muscle-specific AKAP (mAKAP). mAKAP has been shown to compartmentalize to the sarcoplasmic reticulum and perinuclear space of myocytes along with various proteins including: PKA regulatory subunit (RIIa), phosphodiesterase 4D3, protein phosphatase 2A, and ryanodine receptors. Through coordinating spatial-temporal signaling of proteins and enzymes that fine-tunes second messengers, mAKAP functions to regulate cAMP and thus substrate phosphorylation. This leads to changes in cellular Ca2+ availability and Ca2+ sensitivity. We will test the central hypothesis that human mAKAP mutations will modify protein-protein interaction binding kinetics and thus change the ability of this scaffolding protein's interaction network to regulate second messenger dynamics. This will be achieved by three Specific Aims: (1) defining the structure-function relationship between AKAP scaffolding protein and its binding partners by using mAKAP scaffolding protein mutants, (2) determining the spatial-temporal intracellular signaling of mAKAP scaffolding protein interaction network dynamics using sub-cellular targeted biosensors, proteomics and bioinformatics, and (3) determining PKA-dependent substrate phosphorylation and intracellular Ca2+ cycling using adenoviral mediated mutant mAKAP scaffolding proteins expressed in isolated cardiomyocytes. Characterization of mAKAP scaffolding protein-protein interactions will improve our understanding for this central regulator of kinase, phosphodiesterase and phosphatase cardiac intracellular signaling. Overall, this study will (a) advance the understanding of the complexity local versus global signaling of protein interaction networks, (b) train and prepare the next generation of scientists to effectivel confront current and future scientific challenges, theories and paradigms, and (c) serve as the basis toward understanding how perturbations in local signaling of protein interaction networks may be attributed to cardiac diseases, including arrhythmias, hypertrophy, and heart failure.